Nitric oxide donors for inducing neurogenesis

ABSTRACT

There is provided a method of promoting neurogenesis by administering a therapeutic amount of a nitric oxide donor compound to a patient in need of neurogenesis promotion. Also provided is a compound for providing neurogenesis having an effective amount of a nitric oxide donor sufficient to promote neurogenesis. A nitric oxide compound for promoting neurogenesis is also provided. Further, a method of augmenting the production of brain cells and facilitating cellular structural and receptor changes by administering an effective amount of a nitric oxide donor compound to a site in need of augmentation is provided. There is provided a method of increasing both neurological and cognitive function by administering an effective amount of a nitric oxide donor compound to a patient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-In-Part of Ser. No. 10/018,201 filedDec. 14, 2001, which is a National Phase filing of PCT/US/00/16353 filedJun. 14, 2000, which claims the benefit of priority under 35 U.S.C.§119(e) of U.S. Provisional Patent Application Ser. No. 60/138,971,filed Jun. 14, 1999, and which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present application relates to a method and compound for promotingneurogenesis and promoting recovery after neural injury andneurodegeneration. More specifically, the present invention relates to amethod and composition for promoting neurogenesis and plasticity in thenervous system.

2. Description of Related Art

Stroke occurs when a section of the brain becomes infarcted, resultingin death of brain tissue from interruption of cerebral blood supply.Cerebral infarcts associated with acute stroke cause sudden and dramaticneurological impairment. Stroke is the third most common cause of deathin the adult population of the United States, and is a major cause ofdisability.

Pharmacological interventions have attempted to maximize the blood flowto stroke affected brain areas which might be able to survive, butclinical effectiveness has proven elusive. As stated in Harrison'sPrinciples of Internal Medicine (9^(th) Ed., 1980, p. 1926), “despiteexperimental evidence that . . . [cerebral vasodilators] increase thecerebral blood flow, as measured by the nitrous oxide method, they havenot proved beneficial in careful studies in human stroke cases at thestage of transient ischemic attacks, thrombosis-in-evolution, or in theestablished stroke. This is true of nicotinic acid, Priscoline, alcohol,papaverine, and inhalation of 5% carbon dioxide. In opposition to theuse of these methods is the suggestion that vasodilators are harmfulrather than beneficial, since by lowering the systemic blood pressurethey reduce the intracranial anastomotic flow, or by dilating bloodvessels in the normal parts of the brain they steal blood from theinfarct.”

It would therefore be useful to develop a compound and method forlessening the effects of stroke by enabling neurogenesis to occur.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a method ofpromoting neurogenesis by administering a therapeutic amount of a nitricoxide donor to a patient in need of neurogenesis promotion. Neurogenesisis also promoted in non injured brain. Also provided is a compound forinducing neurogenesis including an effective amount of a nitric oxidedonor sufficient to promote neurogenesis. A nitric oxide compound forpromoting neurogenesis is also provided. Further, a method of augmentingthe production of neurons by administering an effective amount of anitric oxide donor compound to a site in need of augmentation isprovided. There is provided a method of increasing both neurological andcognitive function by administering an effective amount of a nitricoxide donor compound to a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings wherein:

FIG. 1 is a photograph showing the BrdU-positive nuclei in the selectedareas;

FIGS. 2A and 2B are graphs showing the amount of BrdU-positive cells inthe subventricular zone (SVZ);

FIG. 3 is a graph showing the amount of BrdU-positive cells in thedentate gyrus;

FIGS. 4A and 4B are graphs showing the percent of distribution of BrdUcells in the dentate gyrus;

FIG. 5 is a photograph showing the size of BrdU immunoreactive cells inrelation to granule cells in granule layers;

FIGS. 6A and 6B are graphs showing the amount of BrdU-positive cells inthe SVZ;

FIGS. 7A and 7B are graphs showing the amount of BrdU-positive cells inthe olfactory bulb (OB);

FIGS. 8A and 8B are graphs showing the amount of BrdU-positive cells inthe dentate gyrus;

FIG. 9 is a graph showing a lesion volume study;

FIG. 10 is a graph showing in Time versus MCAo, the results of anadhesive removal test;

FIG. 11 is a graph showing the results of a Rotarod test;

FIG. 12 is a graph showing the result of the NSS test;

FIG. 13 is a graph showing the percent weight;

FIG. 14 is a graph showing the results of a Rotarod test;

FIG. 15 is a graph showing further results of a Rotarod test

FIG. 16 is a graph showing the results of the footfault test;

FIG. 17 is a graph showing the results of further adhesive removaltests;

FIGS. 18A and 18B are bar graphs showing cell proliferation in DentateGyrus (FIG. 18A) and SVZ (FIG. 18B) in ischemic mice treated with salineand varying doses of sildenafil;

FIGS. 19A-F are photographs and graphs showing TuJ1 immunoreactive cellsin the SVZ (FIGS. 19A-C) and dentate gyrus (FIGS. 19D-F) 28 days afterischemia;

FIGS. 20A and 20B are line graphs showing the effects of sildenafiltreatment on the foot fault test;

FIGS. 21A and 21B are line graphs showing the effects of sildenafiltreatment on the adhesive removal test;

FIGS. 22A and 22B are line graphs showing the effects of sildenafiltreatment on animal body weight loss;

FIGS. 23A-C are line graphs showing the effects of sildenafil treatmenton the foot fault test (FIG. 23A), adhesive removal test (FIG. 23B), andbody weight loss (FIG. 23C) when treatment was initiated 24 hours afterischemia;

FIGS. 24A and 24B are bar graphs showing levels of cGMP in thecerebellum (FIG. 24A) and cortex (FIG. 24B) after treatment withsildeafil in non ischemic rats; and

FIGS. 25A and 25B are photographs showing RT-PCR of PDE5A1 (FIG. 25A)and PDE5A2 (FIG. 25B) mRNA in the cortex of non ischemic rats and theipsilateral cortex of rats 2 hours to 7 days after ischemia.

DETAILED DESCRIPTION OF THE INVENTION

Generally, the present invention provides a method and compound forpromoting neurogenesis. More specifically, the present inventionprovides a method and compound for promoting neurogenesis utilizing aneffective amount of a nitric oxide donor which promotes theneurogenesis. The neurogenesis can be required in various locations,including but not limited to, the brain, CNS, ear or any other locationcontaining damaged neurons therein.

By “nitric oxide donor” it is meant a compound which is able to donatenitric oxide or promote an increase in nitric oxide. There are familiesof compounds which donate nitric oxide. Included among these compoundsare: DETANONOate (DETANONO, NONOate or 1-substituteddiazen-1-ium-1,2-diolates are compounds containing the [N(O)NO]—functional group: DEA/NO; SPER/NO; DETA/NO; OXI/NO; SULFI/NO; PAPA/NO;MAHMA/NO and DPTA/NO), PAPANONOate, SNAP(S-nitroso-N-acetylpenicillamine), sodium nitroprusside, sodiumnitroglycerine, sildenafil (VIAGRA™), and LIPITOR™. There are compoundswhich promote the increase in nitric oxide, such as phosphodiesteraseinhibitors and L-arginine.

By “promoting neurogenesis” as used herein, it is meant that neuralgrowth is promoted or enhanced. This can include, but is not limited to,new neuronal growth or enhanced growth of existing neurons, as well asgrowth and proliferation of parenchymal cells and cells that promotetissue plasticity. Neurogenesis also encompasses, but is not limited to,neurite and dendritic extension and synaptogenesis.

By “augmentation” as used herein, it is meant that growth is eitherenhanced or suppressed as required in the specific situation. Therefore,if additional neuron growth is required, the addition of a nitric oxidedonor increases this growth. Nitric oxide donors, or sources of nitricoxide, prime cerebral tissue to compensate for damage brought on byinjury, neurodegeneration, or aging by enhancing receptor activation andpromoting cellular morphological change and cellular proliferation.

By “neurological” or “cognitive” function as used herein, it is meantthat the neural growth in the brain enhances the patient's ability tothink, function, etc. Humans treated with nitric oxide have increasedproduction of brain cells that facilitate improved cognitive, memory,and motor function. Further, patients suffering from neurologicaldisease or injury, when treated with nitric oxide, have improvedcognitive, memory, and motor function.

The purpose of the present invention is to promote an improved outcomefrom ischemic cerebral injury, or other neuronal injury, by inducingneurogenesis and cellular changes that promote functional improvement.Patients suffer neurological and functional deficits after stroke, CNSinjury, and neurodegenerative disease. These findings provide a means toenhance brain compensatory mechanism to improve function after CNSdamage or degeneration. The induction of neurons and cellular changesinduced by nitric oxide administration promotes functional improvementafter stroke, injury, aging, and degenerative disease. This approach canalso provide benefit to patients suffering from other neurologicaldisease such as, but not limited to, ALS, MS, and Huntingtons

Nitric oxide administered at propitious times after CNS injury promotesneurogenesis in brain and is able to facilitate neurogenesis. Theprimary mechanism for such production is that NO activates glutamatereceptors. These glutamate receptors promote long term potentiation andsubsequently induce regeneration of neurons. As an initial experiment,DETA/NO was employed, a compound with a long half-life (˜50 hours) whichproduces NO. Increased numbers of new neurons were identified when thiscompound was administered at and beyond 24 hours after onset of stroke.

The experimental data included herein show that a pharmacologicalintervention designed to induce production of NO can promoteneurogenesis. Two compounds have been employed, DETANONOate and SNAP,these compounds have successfully induced neurogenesis and improvedfunctional outcome after stroke. The compound used likely crosses theblood brain barrier. Neurogenesis is a major last goal in neuroscienceresearch. Developing a way to promote production of neurons opens up theopportunity to treat a wide variety of neurological disease, CNS injuryand neurodegeneration. It is possible to augment the production ofneurons in non-damaged brain, so as to increase function.

Additionally, the experimental data shows that administration of an NOdonor to rats subjected to stroke significantly increases brain levelsof cGMP, enhances neurogenesis and improves functional recovery (Zhanget al., 2001). Significant functional recovery can be due to increasesin levels of cGMP resulting from administration of an NO donor.Phosphodiesterase type 5 (PDE5) enzyme is highly specific for hydrolysisof cGMP and is involved in regulation of cGMP signaling. Sildenafil isan inhibitor of PDE 5 and causes intracellular accumulation of cGMP. Itis further disclosed that treatment of stroke in the adult rats withVIAGRA™ (content sildenafil) significantly increases numbers ofprogenitor cells and numbers of Tuj1 (a neuronal marker) immunoreactivecells in the ischemic brain, and enhances functional recovery afterstroke.

The market for a class of drugs that promotes the production of neuronsis vast. Nitric oxide donors, of which DETANONO is but one example,promote neurogenesis. Increasing neurogenesis translates into a methodto increase and improve neurological, behavioral, and cognitivefunction, injured because of age, injury, or disease.

The above discussion provides a factual basis for the use of nitricoxide to promote neurogenesis. The methods used with and the utility ofthe present invention can be shown by the following non-limitingexamples and accompanying figures.

Methods:

General methods in molecular biology: Standard molecular biologytechniques known in the art and not specifically described weregenerally followed as in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989),and in Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1989) and in Perbal, A Practical Guideto Molecular Cloning, John Wiley & Sons, New York (1988), and in Watsonet al., Recombinant DNA, Scientific American Books, New York and inBirren et al (eds) Genome Analysis: A Laboratory Manual Series, Vols.1-4 Cold Spring Harbor Laboratory Press, New York (1998) and methodologyas set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531;5,192,659 and 5,272,057 and incorporated herein by reference. Polymerasechain reaction (PCR) was carried out generally as in PCR Protocols: AGuide To Methods And Applications, Academic Press, San Diego, Calif.(1990). In-situ (In-cell) PCR in combination with Flow Cytometry can beused for detection of cells containing specific DNA and mRNA sequences(Testoni et al, 1996, Blood 87:3822.)

General methods in immunology: Standard methods in immunology known inthe art and not specifically described are generally followed as inStites et al. (eds), Basic and Clinical Immunology (8th Edition),Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi (eds),Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York(1980).

Delivery of Therapeutics:

The compound of the present invention is administered and dosed inaccordance with good medical practice, taking into account the clinicalcondition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

In the method of the present invention, the compound of the presentinvention can be administered in various ways. It should be noted thatit can be administered as the compound or as pharmaceutically acceptablesalt and can be administered alone or as an active ingredient incombination with pharmaceutically acceptable carriers, diluents,adjuvants and vehicles. The compounds can be administered orally,subcutaneously or parenterally including intravenous, intraarterial,intramuscular, intraperitoneally, and intranasal administration as wellas intrathecal and infusion techniques. Implants of the compounds arealso useful. The patient being treated is a warm-blooded animal and, inparticular, mammals including man. The pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles as well as implant carriersgenerally refer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention.

It is noted that humans are treated generally longer than the mice orother experimental animals exemplified herein which treatment has alength proportional to the length of the disease process and drugeffectiveness. The doses may be single doses or multiple doses over aperiod of several days, but single doses are preferred.

The doses may be single doses or multiple doses over a period of severaldays. The treatment generally has a length proportional to the length ofthe disease process and drug effectiveness and the patient species beingtreated.

When administering the compound of the present invention parenterally,it will generally be formulated in a unit dosage injectable form(solution, suspension, emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions or dispersionsand sterile powders for reconstitution into sterile injectable solutionsor dispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compoundcompositions. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.According to the present invention, however, any vehicle, diluent, oradditive used would have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various of the other ingredients,as desired.

A pharmacological formulation of the present invention can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicle, adjuvants, additives, anddiluents; or the compounds utilized in the present invention can beadministered parenterally to the patient in the form of slow-releasesubcutaneous implants or targeted delivery systems such as monoclonalantibodies, vectored delivery, iontophoretic, polymer matrices,liposomes, and microspheres. Examples of delivery systems useful in thepresent invention include: U.S. Pat. Nos. 5,225,182; 5,169,383;5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233;4,447,224; 4,439,196; and 4,475,196. Many other such implants, deliverysystems, and modules are well known to those skilled in the art.

A pharmacological formulation of the compound utilized in the presentinvention can be administered orally to the patient. Conventionalmethods such as administering the compounds in tablets, suspensions,solutions, emulsions, capsules, powders, syrups and the like are usable.Known techniques which deliver it orally or intravenously and retain thebiological activity are preferred.

In one embodiment, the compound of the present invention can beadministered initially by intravenous injection to bring blood levels toa suitable level. The patient's levels are then maintained by an oraldosage form, although other forms of administration, dependent upon thepatient's condition and as indicated above, can be used. The quantity tobe administered will vary for the patient being treated and will varyfrom about 100 ng/kg of body weight to 100 mg/kg of body weight per dayand preferably will be from 10 mg/kg to 10 mg/kg per day.

EXAMPLES Example 1

A pharmacological method to promote neurogenesis in brain was developed.Male Wistar rats were subjected to middle cerebral artery (MCA)occlusion by means of intraarterial placement of a clot at the origin ofthe right MCA. Animals were administered (iv/ip) nitric oxide donorcompounds (DETANONO) after induction of stroke, at 24 and 48 hours(Group 1), or at 24 hours followed by daily injection (ip) of NO donorcompound (Group 2). BrdU, a thymidine analog which identifies theformation of new cells, was injected (ip) daily over a period of 14 daysfrom the onset of ischemia. Identification of cell type was determinedby labeling immunoreactivity of specific cell proteins. Thus, neuronswere identified by expression of NeuN, MAP2 and the astrocytes formed byGFAP. Measurements of neurogenesis were performed within specificregions of brain, the subventricular zone and the dentate gyrus.

Results: The data showed a significant increase in the numbers of BrdUpositive cells in rats treated with DETANONO compared to those found inthe untreated group. For Group 2, the results were as follows:subventricular zone: 2748±326 vs. 1653±91.4, dentate gyrus: granule celllayer, 135±18.9 vs. 37.3±3.6; 53.7±6.3 vs. 34.9±2.8, hilus, 43.8±10.2vs. 10.1±2.4. For Group 1, a significant increase in BrdU cells in thegranule cell layer was detected 89.5±12 vs. 37.3±3.6 in treated vs.non-treated rats, respectively. The vast majority of newly formed cells(>90%) within the dentate gyrus were neurons. In other areas of thebrain, newly formed cells had glial and astrocytic phenotype.

Treatment of non-ischemic brain with DETANONO: Rats not subjected to anysurgical procedures were treated with DETANONO. The drug wasadministered as a single dose (iv 0.12 mg). BrdU was injected daily for14 days after treatment. One population (Group 3) of rats was sacrificedon the last day of BrdU injection, Another population (Group 4) wassacrificed at four weeks after the last BrdU injection. Animals whichwere not administered DETA-NONO were given BrdU with the identicalprotocol as that for the DETA-NONO treated rats (Group 5).

Results of Group 3 versus Group 5 were as follows: In the subventricularzones the results were respectively, 2952±102.6 vs. 1432.6±104.6;2725.3±115.5 vs. 1655.2±102.9 in the dentate gyrus (granule cell layer)73.7±8.11 vs. 39.9±7.26. In Group 4 versus Group 5, in thesubventricular zone the results were as follows: 456.5±42.3 vs.214.6±67.9; 518.4±67.2 vs. 233.1±49.2, respectively; in the dentategyrus (hilus) 7.71±89 vs. 3.23±1.22, respectively. Rats treated withDETANONO exhibited a significant increase in newly formed cells, at bothtime points compared to non treated rats. Increases in newly formedcells were apparent in the subventricular zone and in the hippocampus.BrdU reactive cells were double labeled with neuronal markers NeuN andMAP2, and an astrocytic marker, GFAP. Newly formed cells exhibitedneuronal or astrocytic proteins.

FIG. 1 shows double labeling immunohistochemistry within the hippocampusfor BrdU and neuronal markers, NeuN and MAP2, and BrdU with theastrocytic marker, and GFAP in rats treated with DETANONO and subjectedto strokes. Cells exhibited immunoreactivity to both markers, indicatingboth neuronal and astrocytic phenotype of the newly formed cells. It isestimated that more than 90% of the newly formed cells within thehippocampus are neuronal phenotype.

These data indicate that administration of a NO donor promotesneurogenesis in ischemic brain. This approach is applicable to manyforms of CNS pathology and injury. In addition, NO also promotesneurogenesis in “normal” adult brain.

The purpose of the present invention is to promote an improved outcomefrom ischemic cerebral injury, or other neuronal injury, by inducingneurogenesis. Patients suffer neurological and functional deficits afterstroke, CNS injury and neurodegenerative disease. These findings providea means to enhance brain compensatory mechanism to improve functionafter CNS damage or degeneration. The induction of neurons will promotefunctional improvement after stroke.

Nitric oxide administered at propitious times after CNS injury promotesneurogenesis in brain and is able to facilitate neurogenesis. Themechanism for such production is that NO activates glutamate receptors.These glutamate receptors promote long term potentiation andsubsequently induce regeneration of neurons. As an initial experiment,DETA/NO was employed, a compound with a long half-life (˜50 hours) whichproduces NO. Increased numbers of new neurons were identified when thiscompound was administered at and beyond 24 hours after onset of stroke.

The experimental data included herein show that a pharmacologicalintervention designed to induce production of NO can promoteneurogenesis. The compound used likely crosses the blood brain barrier.Neurogenesis is a major last goal in neuroscience research. Developing away to promote production of neurons opens up the opportunity to treat awide variety of neurological disease, CNS injury and neurodegeneration.It is possible to augment the production of neurons in non-damagedbrain, so as to increase function.

The market for a class of drugs that promotes the production of neuronsis vast. Nitric oxide donors, of which DETANONO is but one example,promote neurogenesis. Increasing neurogenesis translates into a methodto increase, improve neurological, behavioral and cognitive function,with age and after injury or disease.

There have previously been no applications of NO donors, or this drug inparticular, to the induction of neurogenesis after stroke.

Adult rodent brain is capable of generating neuronal progenitor cells inthe subventricular zone (SVZ) and in the dentate gyrus of thehippocampus throughout the life of the animal. However, signals thatregulate progenitor cell proliferation and differentiation are notknown. Nitric oxide (NO) is a chemical messenger in biological systemsand serves as a neurotransmitter in the brain. In the present study, theeffects of NO on the proliferation of neuronal progenitor cells in theSVZ and in the dentate gyrus of adult rats was explored.

Two experiments were performed. In the first experiment, the effects ofNO on the proliferation of neuronal progenitor cells in the SVZ and thedentate gyrus of non ischemic adult rats were examined. In the secondexperiment, the effects of NO on the proliferation of neuronalprogenitor cells in the SVZ and in the dentate gyrus of ischemic adultrats were examined.

Male Wistar rats weighing 300-350 g were used in the present studies(Charles River Breeding Company, Wilmington, Mass.). DETANONOate, an NOdonor with a half-life 20 hours under physiological conditions, waspurchased from ALEXIS Biochemical Corporation. Bromodeoxyuridine (BrdU),the thymidine analog used as mitotic labeling, was purchased from SigmaChemical. A mouse monoclonal antibody against BrdU was purchased fromBoehringer Mannheim.

Male Wistar rats (n=28) weighing 300-350 g were anesthetized withhalothane (1-3.5% in a mixture of 70% N₂O and 30% O₂) using a face mask.The rectal temperature was maintained at 37±1° C. throughout thesurgical procedure using a feedback regulated water heating system. Theright femoral artery and vein were cannulated with a PE-50 catheter forcontinuous monitoring of blood pressure and measurement of blood gases(pH, pO₂, pCO₂) and for drug administration, respectively. DETANONOatewas intravenously and intraperitoneally injected to rats.

DETANONO treatment: Rats were randomly divided into four groups. Group 1(single Rx), rats were intravenously injected with four consecutivebolus doses of DETANONO (0.1 mg/kg each) every fifteen minutes (totaldose of 0.4 mg/kg). Group 2 (two Rx group), rats were intravenouslyinjected with four consecutive bolus doses of DETANONO (0.1 mg/kg each)every fifteen minutes (total dose 0.4 mg/kg) and rats received a secondtreatment at 24 hours later. Group 3 (seven Rx group), rats receivedfour consecutive intravenous bolus doses of DETANONO (0.1 mg/kg each)every fifteen minutes on the first experimental day and rats wereintraperitoneally injected with a bolus dose of DETANONO (0.4 mg/kg)daily for an additional six consecutive days. Group 4 (control), ratsreceived saline only (single dose).

Rats received an intraperitoneal injection of BrdU (50 mg/kg) on thefirst day of DETANONO treatment and daily intraperitoneal injections ofBrdU for fourteen consecutive days. To determine whether theproliferation and the differentiation of cells in the SVZ and dentategyrus of adult rats is affected by NO, rats were sacrificed at fourteendays (n=3-5 per group) and 42 days (n=3-5 per group) after first dose ofDETANONO treatment, respectively. Rats were transcardially perfused with4% paraformaldehyde in 100 mM phosphate buffer, pH 7.4. Brains wereremoved and fixed in 4% formaldehyde.

For BrdU immunostaining, DNA was first denatured by incubating brainsections (6 μm) in 50% formamide 2×SSC at 65° C. for 2 hours and then in2N HCl at 37° C. for 30 minutes. Sections were then rinsed with Trisbuffer and treated with 1% of H₂O₂ to block endogenous peroxidase.Sections were incubated with a primary antibody to BrdU (1:100) at roomtemperature for one hour and then incubated with biotinylated secondaryantibody (1:200, Vector, Burlingame, Calif.) for one hour. Reactionproduct was detected using 3′3′-diaminobenzidine-tetrahydrochloride(DAB, Sigma).

BrdU immunostained sections were digitized under 40× objectively(Olympus BX40) via the MCID computer imaging analysis system (ImagingResearch, St. Catharines, Canada). BrdU immunoreactive nuclei werecounted on a computer monitor to improve visualization and in one focalplane to avoid over-sampling. Structures were sampled either byselecting predetermined areas on each section (RMS and OB) or byanalyzing entire structures on each section (dentate gyrus and SVZ).

Every 40^(th) coronal section was selected from each rat for a total ofseven sections between AP+10.6 mm, genu corpus callosum, and AP+8.74mm-anterior commissure crossing (Paxinos and Watson, 1986). BrdUimmunoreactive-positive nuclei were counted in the lateral ventriclewall. All BrdU immunoreactive-positive nuclei in these areas arepresented as the number of the BrdU immunoreactive cells/mm². Densityfor the seven sections was averaged to obtain a mean density value foreach animal.

Every 20^(th) section was selected from each rat for a total of sixsections from the sagittal series of the OB/frontal cortex. As depictedin FIG. 1, two predetermined areas (100×100 μm) in the RMS and fourareas (300×300 μm) in the granule cell layer (GCL) of the OB wereanalyzed on each section. All BrdU positive nuclei in these selectedareas are presented as the number of the cells/mm². BrdU density for thesix sections was averaged to obtain a mean density value for eachanimal.

Each 50^(th) coronal section was selected from each rat for a total ofeight sections between AP+5.86 mm and AP+2.96 mm including the hilus,subgranular zone (SGZ), and inner first, second and third of the granulecell layer (GCL). The SGZ, defined as a two-cell body wide zone alongthe border of the GCL and the hilus, were always combined with the GCLfor quantification. All BrdU immunoreactive nuclei in these areas arepresented as the number of the BrdU immunoreactive cells/mm². Densityfor the eight sections was averaged to obtain a mean density value foreach animal.

Results

Rats treated with DEANONO have a significant (p<0.05) increase innumbers of BrdU immunoreactive cells in the SVZ compared with ratstreated with saline at fourteen days and 42 days after treatment (FIG. 2a). Rats that received seven doses of DETANONO exhibited the highestnumber of BrdU immunoreactive cells compared with rats that received oneand two doses of DETANONO at fourteen days after treatment. There was asignificant difference in numbers of BrdU immunoreactive cells betweenone dose and seven doses of DETANONO was detected (FIG. 2 b), suggestingthat increases in BrdU immunoreactive cells is dose dependent manner.Although numbers of BrdU immunoreactive cells decreased at 42 days aftertreatment as compared with the number of cells at 14 days, the number ofBrdU immunoreactive cells remained significantly increased compared withthe number in control saline animals (FIG. 2 a).

Numbers of BrdU immunoreactive cells did not significantly increase inthe RMS in rats treated with DETANONO at fourteen days and 42 days aftertreatment (Table 1). However, significant increases in BrdUimmunoreactive cells were detected in OB at 42 days after a single setof DETANONO treatment and at fourteen days and 42 days after two andseven sets of DETANONO treatment compared with the control group (Table1), suggesting an increased migration of SVZ progenitors.

A single DETANONO treatment did not significantly increase in numbers ofBrdU immunoreactive cells in the dentate gyrus at fourteen days and 42days after treatment (FIG. 3). In contrast, rats treated with two andseven sets of DETANONO exhibited significant (p<0.01) increases innumbers of BrdU immunoreactive cells in the dentate gyrus at fourteendays and 42 days after treatment compared with the control group (FIG.3). Percentage of distribution of BrdU immunoreactive cells in thedentate gyrus showed that treatment with DETANONO significantlydecreases percentage of BrdU immunoreactive cells in subgranular zoneand significantly increases in the granule layers at fourteen days and42 days after treatment compared with the control group (FIGS. 4 a and 4b), indicating that NO promote migration of BrdU immunoreactive cells.The BrdU immunoreactive cells were oval and rounded and either the samesize or smaller than nuclei of the granule cells in granule layers (FIG.5).

The data demonstrate that treatment with DETANONO to adult rats not onlyincreases proliferation of SVZ and dentate gyrus progenitor cells butalso prolongs survival of proliferated progenitor cells. Some BrdUimmunoreactive cells have morphological characteristics of granule cellsin the dentate gyrus. Thus, the data suggest that NO enhancesneurogenesis in adult rat brain.

Based on above data, a second experiment was performed to explore NOeffects on focal embolic cerebral ischemic brain. All procedures werethe same as in the first experiment except for the following procedures.

Male Wistar rats (n=30) weighing 300-350 g were subjected to middlecerebral artery (MCA) occlusion. The MCA was occluded by placement of anembolus at the origin of the MCA. Briefly, a single intact fibrin rich24 hour old homologous clot (about 1 μl) was placed at the origin of theMCA via a fifteen mm length of modified PE-50 catheter. The experimentalprotocol consisted of four groups. In Group I (control group), rats weresubjected to MCA occlusion and received four consecutive intravenousbolus doses of saline (0.52 ml each, every fifteen minutes) at 24 hoursafter ischemia. Group II (DETNO/NO precondition) rats received fourconsecutive intravenous bolus doses of DETANONO (0.1 mg/kg each, everyfifteen minutes, and total dose 0.4 mg/kg) at 24 hours beforeembolization. Group III (DETANONO two set group), animals receivedconsecutive intravenous bolus doses of DETANONO (0.1 mg/kg each, everyfifteen minutes and total dose 0.4 mg/kg) at 24 and 48 hours afterocclusion. Group IV (DETANONO seven set group), animals received fourconsecutive intravenous bolus doses of DETANONO (0.1 mg/kg each, everyfifteen minutes and total dose 0.4 mg/kg) at 24 hours afterembolization. Subsequently, rats were intraperitoneally injected withDETA/NO at 0.4 mg/kg every day for six consecutive days.

Embolic MCA occlusion resulted in significant (p<0.05) increases innumbers of BrdU immunoreactive cells in the ipsilateral SVZ and OB atfourteen days after MCA occlusion compared with non-ischemic rats (Table2). The numbers of BrdU reactive cells decreased at 42 days after MCAocclusion, showing that focal cerebral ischemia induces transientincreases in proliferation of progenitor cells in the ipsilateral SVZ(Table 2). MCA occlusion did not affect proliferation of progenitorcells in the contralateral SVZ and OB and in the both dentate gyrus(Table 2).

Significant (p<0.05) increases in numbers of BrdU immunoreactive cellswere detected in the contralateral SVZ at 14 days after MCA occlusionand in both SVZs at 42 days after MCA occlusion in the preconditionedgroup compared with the non-treated MCA occlusion group (FIGS. 6A, 6B).Rats in two dosage groups had a significant increase in numbers of BrdUimmunoreactive cells in the ipsilateral SVZ at 14 days and also hadsignificant increases in numbers of BrdU immunoreactive cells in bothSVZ at 42 days after MCA occlusion (FIGS. 6A, 6B). Rats treated withseven sets of DETANONO injection exhibited significant increases in BrdUimmunoreactive cells in the contralateral and in the ipsilateral SVZ at14 days and 42 days after MCA occlusion.

Significant increases in BrdU immunoreactive cells were detected in theOB in the ischemic rats treated with DETANONO at 14 days and 42 daysafter MCA occlusion (FIGS. 7A, 7B).

The ischemic rats treated with DETANONO had significant increases inBrdU immunoreactive cells in dentate gyrus at 14 days and 42 days afterMCA occlusion compared with MCA occlusion group (FIGS. 8A, 8B).

The ischemic rats treated with DETANONO did not exhibit a significantreduction of ischemic lesion volume (FIG. 9).

These data demonstrate that embolic MCA occlusion itself increasesproliferation of progenitor cells in the ipsilateral SVZ. Many cellsborn in the SVZ migrate along RMS into the OB, where they differentiateinto neurons. Thus, increases in the number of BrdU immunoreactive cellsin the ipsilateral OB suggest an increased migration of the ipsilateralSVZ progenitor cells. These data also suggest that signals whichincrease proliferation of progenitor cells are transient and local afterMCA occlusion. However, significant increases in proliferation ofprogenitor cells was sustained at least for 42 days after MCA occlusionwhen the ischemic rats were treated with DETANONO. Increases inproliferation of progenitor cells are induced by NO, since increases innumbers of BrdU immunoreactive cells involved not only both SVZ but alsoboth dentate gyrus. However, there are differences in a number of BrdUcells between non-ischemic rats treated with DETANONO and the ischemicrats treated with DETANONO. The ischemic rats treated with DETANONO hadhigher absolute numbers of BrdU immunoreactive cells in the dentategyrus at 14 days and 42 days after MCA occlusion than the numbers ofnon-ischemic rats treated with DETANONO, suggesting that NO may amplifysignals generated by ischemia to increase proliferation of progenitorcells. Therefore, the data indicate that focal cerebral ischemiaproduces transient proliferation of progenitor cells and that NOenhances proliferation of progenitor cells in the ischemic brain.

Example 2

Administration of nitric oxide donor compound (DETA/NO) to normal andischemic rats promotes neurogenesis in non-ischemic and ischemic brains.Since then, additional experiments have been performed to test thehypothesis that neurogenesis induced by DETA/NO promotes functionalimprovement after stroke; the data is provided herewith. Animals wereadministered (iv/ip) DETA/NO at one day (Group 1) or seven days (Group2) after induction of stroke and followed by daily injection (ip) ofDETA/NO over a period of seven days. Another NO donor compound (SNAP)was administered (iv) to ischemic rats at one day and two days afterstroke (Group 3). Young (3 month old) rats were used in Groups 1 and 2.Middle aged rats (10 to 12 months old) were used in Group 3. A batteryof neurological functional tests were measured from two days toforty-two days after stroke. These tests included 1) Neurologicalseverity score (NSS) which measures motor, sensory and reflex functionsand is similar to the contralateral neglect testing described in humans.The higher the score, the more severe the injury; 2) Rotarod testmeasures fore and hindlimb motor coordination and balance. Data arepresented as percentage of baseline values; 3) Footfault test measuresfore and hindlimb motor coordination. The higher the number, the moresevere the injury; 4) Adhesive removal test measures sensorimotorimpairments. Data are presented as time (seconds). The longer the timeperiod, the more severe the injury; 5) Animal body weight.

Results

Group 1: Significant improvements on motor and sensorimotor functions(FIG. 10 Rotarod test, FIG. 11 Adhesive Removal test, FIG. 12 NSS test)and animal body weight (FIG. 13) were detected in rats treated withDETA/NO compared with control rats.

Group 2: A significant improvement of neurological function was onlydetected in Rotarod test at 28 and 42 days after stroke (FIG. 14)compared with control animals.

Group 3: Animals treated with SNAP exhibited significant improvements onmotor and sensorimotor functions (FIG. 15 Rotarod test, FIG. 16footfault test, and FIG. 17 Adhesive removal test) compared with controlanimals.

Conclusions

These data indicate 1) administration of DETANONO to ischemic ratsimproves neurological functional recovery and these improvements can beachieved even when DETANONO is administered seven days after stroke; 2)in addition to DETANONO, administration of SNAP to ischemic rats alsoimproves neurological functions, suggesting that administration of NOdonor compounds can enhance functional recovery; and 3) administrationof SNAP to middle aged rats is effective to promote functional recovery,which is important and clinically relevant because most stroke patientsare middle age and older. These data, together with previous datashowing that NO donor promotes neurogenesis, suggest that NO donorcompounds enhance neurological functional recovery after stroke viapromotion of neurogenesis in ischemic brain.

Example 3

It was found that treatment of focal cerebral ischemia in the adult ratswith VIAGRA™, sildenafil, at a dose 2 or 5 mg/kg significantly improvesrecovery of neurological outcome. Enhanced functional improvements aftertreatment with VIAGRA™ are attributed to brain plasticity because: 1)the treatment did not reduce infarct volume and the treatment waseffective even when treatment was initiated at 24 hours of the onset ofischemia, which is far beyond the therapeutic window forneuroprotection; 2) the treatment significantly increased numbers ofprogenitor cells in the dentate gyrus and subventricular zone (SVZ) aswell as numbers of TuJ1 immuoreactive cells. Furthermore, RT-PCR datarevealed the presence of PDE5 in rat brain and administration of VIAGRA™significantly increased cortical levels cGMP in rats, indicating thateffects of VIAGRA™ on neurological outcome can be mediated by theendogenous PDE5/cGMP pathway.

Materials and Methods

Male Wistar rats weighing 300-350 g were used in the present studies(Charles River Breeding Company. Wilmington, Mass.). A film tablet ofVIAGRA™ (content 100 mg sildenafil) was purchased commercially.Bromodeoxyuridine (BrdU), the thymidine analog used for mitoticlabeling, was purchased from Sigma Chemical. Mouse monoclonal antibodiesagainst BrdU and neuronal class III β-tubulin (TuJ1) was purchased fromBoehringer Mannheim and Covance, respectively.

General Preparation:

Male Wistar rats weighing 300-350 g were anesthetized with halothane(1-3.5% in a mixture of 70% N₂O and 30% O₂) using a face mask. Therectal temperature was maintained at 37±1° C. throughout the surgicalprocedure using a feedback regulated water heating system.

Animal Model:

The MCA was occluded by placement of an embolus at the origin of theMCA.

Immunohistochemistry:

For BrdU immunostaining, DNA was first denatured by incubating brainsections (6 μm) in 50% formamide 2×SSC at 65° C. for 2 hours and then in2 N HCL at 37° C. for 30 minutes. Sections were then rinsed with Trisbuffer and treated with 1% of H₂O₂ to block endogenous peroxidase.Sections were incubated with a primary antibody to BrdU (1:100) at roomtemperature for 1 hour and then incubated with biotinylated secondaryantibody (1:200, Vector, Burlingame, Calif.) for 1 hour. Reactionproducts were detected using 3′3′-diaminobenzidine-tetrahydrochloride(DAB, Sigma). For TuJ1 immunostaining, coronal sections were incubatedwith the antibody against TuJ1 (1:1000) at 4° C. overnight and thenincubated with a biotinylated horse anti-mouse immunoglobulin antibodyat room temperature for 30 minutes. Reaction products were detected withDAB.

cGMP Measurement in Brain Tissue:

Levels of cGMP were measured in non ischemic rat brain. cGMP wasdetermined by a commercially available low pH Immunoassay kit (R&Dsystems Inc, Minneapolis, Minn.). The sensitivity of the assay wasapproximately 0.6 pmol/ml for the non-acetylated procedure. The brainwas rapidly removed and the cortex and the cerebellum were separated.The brain tissue was weighed and homogenized in 10 volume of 0.1 N HClcontaining 1 mM 3-isobutyl-1-methylxanthine (IBMX).

Quantification:

BrdU immunostained sections were digitized using a 40× objective(Olympus BX40) via the MCID computer imaging analysis system (ImagingResearch, St. Catharines, Canada). BrdU immunoreactive nuclei werecounted on a computer monitor to improve visualization and in one focalplane to avoid over-sampling. Structures were sampled by analyzingentire structures on each section (SVZ and dentate gyrus). All BrdUimmunoreactive-positive nuclei in these areas are presented as thenumber of the BrdU immunoreactive cells/mm² and data shown are mean±SE.Density for the selected several sections was averaged to obtain a meandensity value for each animal. For measurements of TuJ1immunoreactivity, a threshold was applied to each digitized image(628×480 μm²) for ensuring that the numbers of TuJ1 immunostained pixelswere representative of the original TuJ1 immunoreactive patterns. Allobjects with fewer than 5 pixels were eliminated from measurements. Dataare presented as a percentage of area, in which the number of TuJ1immunostained pixels was divided by the total number of pixels in thearea (628×480 μm²).

Experimental Protocol:

To examine the effects of VIAGRA™ on cell proliferation, VIAGRA™ at adose of 2 mg/kg (n=10) or 15 mg/kg (n=9) was randomly administeredorally to rats 2 hours after MCA occlusion and daily for an additional 6consecutive days. An additional group of ischemic rats (n=9) was treatedorally with VIAGRA™ (2 mg/kg) 24 hours after MCA occlusion and daily foran additional 6 consecutive days. The ischemic rats (n=9) were treatedwith the same volume of saline as a control group. Rats receivedintraperitoneal injection of BrdU (50 mg/kg) 24 hours after ischemia anddaily intraperitoneal injection of BrdU for 14 consecutive days. Allrats were sacrificed 28 days after ischemia.

To examine whether administration of VIAGRA™ affects neurologicalbehavior, an array of behavior tests (foot-fault and adhesive removetests) and animal body weight were measured in rats of each groupdescribed above at 1.5 hours, 1, 2, 4, 7, 14, 21, and 28 days of theonset of MCA occlusion.

To examine the effects of VIAGRA™ on neurons, TuJ1 immunoreactivity wasmeasured 28 days after ischemia.

To examine whether administration of VIAGRA™ affects brain cGMP levels,non ischemic rats were treated with VIAGRA™ at 2 mg/kg (n=6), 5 mg/kg(n=6) or saline (n=10) for 6 consecutive days. These rats weresacrificed one hour after the last treatment for measurement of braincGMP. cGMP was determined by a commercially available low pH Immunoassaykit (R&D systems Inc, Minneapolis, Minn.).

To examine brain PDE5 in rats, non ischemic rats (n=3) and ischemic ratsat 2, 24, 48, 72, 168 hours of the onset of ischemia (n=3 for each timepoint) were sacrificed. Reverse transcription (RT)-polymerase chainreaction (PCR) was performed to detect PDE5 in brain tissue. The primersamplify a cDNA fragment coding for N-terminal regions of rat PDE5A1(Zhang et al., 2001): the 5′ primer5′-AAAACTCGAGCAGAAACCCGCGGCAAACACC-3′ and the 3′ primer5′-GCATGAGGACTTTGAGGCAGAGAGC-3′. The primers amplify a cDNA fragmentcoding to rat PDE5A2 (Kotera et al., 2000): the 5′ primer5′-ACCTCTGCTATGTTGCCCTTTGC-3′ and the 3′ primer5′-GCATGAGGACTTTGAGGCAGAGAGC-3′.

Results: Effects of VIAGRA™ on Cell Proliferation:

Ischemic rats treated with oral administration of VIAGRA™ (2 or 5 mg/kg)initiated at 2 or 24 hours after stroke had significant (p<0.05)increases in numbers of BrdU immunoreactive cells in the dentate gyrusof both hemispheres (FIG. 18A) while the treatment with VIAGRA™ at dosesof 2 and 5 mg/kg significantly (p<0.05) increased numbers of BrdUimmunoreactive cells in the ipsilateral SVZ and in the SVZ of bothhemispheres (FIG. 18B), respectively, compared with numbers in ratstreated with saline 28 days after ischemia.

More specifically, FIGS. 18A and B are bar graphs which show theproliferating cells in the dentate gyrus (FIG. 18A) and in the SVZ (FIG.18B) in ischemic treated with saline and with different doses ofVIAGRA™. Numbers of 2 and 5 mg in the figure represent 2 and 5 mg/kg ofVIAGRA™ and 2 hours and 24 hours represent the time points whentreatment was initiated. *p<0.05 and **p<0.01 versus the saline treatedgroup.

Effects of VIAGRA™ on Neurons:

Administration of VIAGRA™ at doses of 2 or 5 mg/kg significantly(p<0.05) increased TuJ1 immunoreactive cells in the ipsilateral SVZ(FIGS. 19A to 19C) and striatum (FIGS. 19D to 19F) compared withhomologous tissue in the contralateral hemisphere and with theipsilateral SVZ and striatum of iscehmic rats treated with saline.Clusters of TuJ1 immunoreactive cells were present in the both ofipsilateral SVZ and striatum of VIAGRA™ treated rats (FIGS. 19A and19D).

More specifically, FIG. 19 shows TuJ1 immunoreactive cells in the SVZ(FIGS. 19A to 19C) and dentate gyrus (FIGS. 19D to 19F) 28 days afterischemia. FIGS. 19A and 19D show increases in TuJ1 immunoreactive cellsin the ipsilateral SVZ and the dentate gyrus, respectively, as comparedto their homologous tissue in the contralateral hemisphere (FIGS. 19Band 19E) from a representative rat. FIGS. 19C and 19F show quantitativedata wherein LV is the lateral ventricle and the Bar equals 50 μm.

Effects of VIAGRA™ on Neurological Outcome:

The ischemic rats treated with VIAGRA™ at dose of 2 mg/kg (FIGS. 20A and21A) or 5 mg/kg (FIGS. 20B and 21B) significantly improved performanceon the foot-fault test (FIG. 20) and the adhesive removal test (FIG. 21)during 2 to 21 days compared with the saline treated rats when treatmentwas initiated at 2 hours of the onset of ischemia. In addition,treatment with VIAGRA™ at dose of 2 mg/kg (FIG. 22A) or 5 mg/kg (FIG.22B) significantly reduced animal body weight loss (FIG. 22). Incontrast, infarct volumes measured 28 days after ischemia were notsignificantly different among these groups (Table 3), showing thatinfarct volume dose not contribute to improvement of functionalrecovery. VIAGRA™ was also administered at a dose of 2 mg/kg to theischemic rats starting at 24 hours after onset of ischemia. Althoughmarked neurological impairments were detected one day after ischemia,the ischemic rats receiving VIAGRA™ exhibited significant (p<0.05)improvements on the foot-fault (FIG. 23A) and adhesive removal (FIG.23B) tests during 7 to 28 days. Rats treated with VIAGRA™ also showedsignificant (p<0.05) reduction in body weight loss at 4, 7 14, 21 and 28days after ischemia (FIG. 23C). However, there were no significantdifferences of infarct volume between ischemic animals treated withVIAGRA™ and animals in the control group 28 days after ischemia (Table3).

FIGS. 20A and 20B are line graphs which show the effects of VIAGRA™treatment on the foot fault test (FIG. 20A, 2 mg/kg, and FIG. 20B, 5mg/kg).

FIGS. 21A and 21B are line graphs which show the effects of VIAGRA™treatment on the adhesive removal test (FIG. 21A, 2 mg/kg, and FIG. 21B,5 mg/kg).

FIGS. 22A and 22B are line graphs which show the effects of VIAGRA™treatment on animal body weight loss (FIG. 22A, 2 mg/kg, and FIG. 22B, 5mg/kg).

FIGS. 23A and 23B are line graphs which show the effects of VIAGRA™ (2mg/kg) treatment on the foot-fault test (FIG. 23A), adhesive removaltest (FIG. 23B) and body weight loss (FIG. 23C) when treatment wasinitiated 24 hours after ischemia.

Effects of VIAGRA™ on cGMP:

The cerebellar levels of cGMP (FIG. 24A, saline) were higher than thecortical (FIG. 24B, saline) levels in non ischemic control rats, whichis consistent with previous studies (Kotera et al., 2000). Treatmentwith VIAGRA™ at a dose of 2 or 5 mg/kg for 7 days significantly (p<0.05)increased the cortical (FIG. 24B) but not the cerebellar (FIG. 24A)levels of cGMP compared with levels in the control group.

FIGS. 24A and 24B are bar graphs which show levels of cGMP in thecerebellum (FIG. 24A) and cortex (FIG. 24B) after treatment with VIAGRA™in non ischemic rats.

PDE5 in Rat Brain:

RT-PCR analysis revealed both PDE5A1 (FIG. 25, 257 bp) and PDE5A2 (FIG.25, 149 bp) transcripts were present in non ischemic rat brain tissue,indicating the presence of PDE5. MCA occlusion did not change levels ofPDE5A1 and PDE5A2 compared with levels in non ischemic rats (FIG. 25).

FIGS. 25A and 25B are photographs showing RT-PCR of PDE5A1 (FIG. 25A)and PDE5A2 (FIG. 25B) mRNA in the cortex of non ischemic rats (N in FIG.25A and FIG. 25B) and the ipsilateral cortex of rats 2 hours to 7 daysafter ischemia, wherein M=marker, N=non ischemic rats, 2 hours, 4 hours,1 day, 2 days, and 7 days are the times after ischemia.

Throughout this application, various publications, including UnitedStates patents, are referenced by author and year and patents by number.Full citations for the publications are listed below. The disclosures ofthese publications and patents in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the described invention, theinvention may be practiced otherwise than as specifically described.

TABLE 1 Density of newborn cells in the brain DETA NO DETA NO DETA NO ×Area Saline ONCE TWICE 7 TIMES Rostral migratory stream (BrdU 50 mg/kgip daily × 14 d) 1 day after last BrdU injection (14 d) Right side 869.2± 98.25 950.25 ± 99.55    991 ± 98.25 1169.4 ± 218.85 1 day after lastBrdU injection (14 d) Left side 841.9 ± 230.4 998.55 ± 59.7  1070.85 ±160.1   1312.5 ± 265   4 weeks after last BrdU injection Right side21.85 ± 6.55  22.5 ± 5.95 32.9 ± 8.15   45 ± 11.35 4 weeks after lastBrdU injection Left side 21.2 ± 5.2  26.25 ± 6.9  37.8 ± 5.15 47.5 ±15.6 Olfactory bulb (BrdU 50 mg/kg ip daily × 14 d) 1 day after lastBrdU injection (14 d) Ipsilateral 45.15 ± 7.4  41.4 ± 5.55  91.65 ±12.35* 106.25 ± 17.7** 1 day after last BrdU injection (14 d)Contralateral 31.55 ± 8.45  39.75 ± 6.2    99.6 ± 10.5**  116.55 ±16.45** 4 weeks after last BrdU injection (42 d) Ipsilateral 12.95 ±2.6    75.65 ± 10.85**      85 ± 15.95**  84.4 ± 7.1** 4 weeks afterlast BrdU injection (42 d) Contralateral 9.95 ± 2.85     80 ± 12.3**   98.4 ± 19.95** 100.65 ± 19**   Densitites of newborn cells arepresented as the mean number of BrdU-positive cells per mm² ± SEM.Values different from saline treatment group, *p < 0.05, **p < 0.01.

TABLE 2 Density of newborn cells in the brain Area Ischemia Only Nolschemia Subventricular zone (BrdU 50 mg/kg ip daily × 14 d) 1 day afterlast BrdU injection (14 d) Right side  3237.77

 179.14** 2301.64

 171.37  1 day after last BrdU injection (14 d) Left side 2361.49

 156.55  2094.06

 229.20  4 weeks after last BrdU injection Right side 272.96

 32.66  222.07

 21.81  4 weeks after last BrdU injection Left side 206.16

 13.00  191.86

 15.88  Rostral migratory stream (BrdU 50 mg/kg ip daily × 14 d) 1 dayafter last BrdU injection (14 d) Right side   1185

 197.65 869.2

 98.25 1 day after last BrdU injection (14 d) Left side 1008.75

 137.1  841.9

 230.4 4 weeks after last BrdU injection Right side 38.15

 20.65 21.85

 6.55  4 weeks after last BrdU injection Left side 18.75

 7.2  21.2

 5.2  Olfactory bulb (BrdU 50 mg/kg ip daily × 14 d) 1 day after lastBrdU injection (14 d) Right side  90.7

 8.6** 45.15

 7.4  1 day after last BrdU injection (14 d) Left side 48.45

 5.9  31.55

 8.45  4 weeks after last BrdU injection (42 d) Right side 11.4

 1.45 12.95

 2.6  4 weeks after last BrdU injection (42 d) Left side 8.85

 0.95 9.95

 2.85 Dentate gyrus (BrdU 50 mg/kg ip daily × 14 d) 1 day after lastBrdU injection (14 d) Right side 55.11

 4.06  61.31

 4.49  1 day after last BrdU injection (14 d) Left side 57.00

 3.99  64.44

 4.13  4 weeks after last BrdU injection (42 d) Right side 30.20

 4.81  36.99

 2.73  4 weeks after last BrdU injection (42 d) Left side 29.80

 4.32  40.33

 3.72  Densitites of newborn cells are presented as the mean number ofBrdU-positive cells per mm² ± SEM. Values different from non ischemicgroup, *p < 0.05, **p < 0.01.

TABLE 3 Infarct volume % of infarct volume at Groups Treatment StartDoses 28 days (Mean ± SE) Viagra group 1  2 h after MCAo 2 mg/kg oral ×7days 35.15 ± 3.25 Viagra group 2  2 h after MCAo 5 mg/kg oral × 7days37.67 ± 4.33 Viagra group 3 24 h after MCAo 2 mg/kg oral × 7days 35.52 ±0.93 Contrao (Saline)  2 h after MCAo Saline oral × 7 days 38.32 ± 1.74

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1. A method of promoting neurogenesis comprising the step of: administering a therapeutic amount of a nitric oxide donor compound to a patient in need of neurogenesis promotion.
 2. A compound for promoting neurogenesis comprising an effective amount of a nitric oxide donor sufficient to promote neurogenesis.
 3. A neurogenesis promoter comprising a nitric oxide donor in a pharmaceutically acceptable carrier.
 4. The neurogenesis promoter according to claim 3, wherein said nitric oxide donor augments nitric oxide in a tissue.
 5. The neurogenesis promoter according to claim 4, wherein said nitric oxide donor is selected from the group consisting essentially of phosphodiesterase inhibitors, L-arginine, sildenafil, and LIPITOR.
 6. A method of augmenting the production of neurons by administering an effective amount of a nitric oxide donor to a site in need of augmentation.
 7. A method of increasing neurological function by administering an effective amount of a nitric oxide donor to a patient.
 8. A method of increasing cognitive and neurological function by administering an effective amount of a nitric oxide donor compound to a patient. 